In this project we focus on epigenetic and signal-transduction mechanisms that regulate cell-specific OT and VP gene expression in the hypothalamus. In addition we do experiments in this project which continue to refine the organotypic culture of hypothalamus model that we previously developed in our laboratory, and which has proven to be so valuable for our signal-transduction mechanisms studies in the SCN (Rusnak et. al., 2007). A key issue in this project is to evaluate the chromatin status of the OT and VP genes in the SON. Our hypothesis is that certain chromatin modifications in the two genes are specifically expressed in the OT and VP neuronal phenotypes. One specific epigenetic mechanism that we attempted to study is the acetylation and methylation of histones on the OT and VP genes in the OT- and VP-MCNs. This type of chromatin modification is very dynamic &therefore reversible, so the conditional co-expression of OT &VP observed in some MCNs in the SON could occur under some circumstances (see Glasgow et al, 1999;Xi et al, 1999). To address this hypothesis, we did various epigenetic experiments focused on analyzing specific histone acetylation &methylation patterns in OT &VP chromatin in SON and in other specific genes, under various physiological conditions in vivo, and tested effects of acetylation/deacetylation inhibitors on OT &VP chromatin and hnRNA transcription in vivo. Unfortunately, these experiments were not successful, in large part, because the harvesting of SON tissues for the isolation of chromatin by conventional tissue punch techniques produced too much contaminating chromatin from non-MCN cells thereby creating an unfavorable signal to noise situation for the subsequent ChIP methodology. Similar experiments will be attempted again in the coming year, but will use laser microdissected (LCM) MCNs from the SON in an effort to improve the signal to noise situation for the ChIP protocol. The second specific epigenetic mechanism that we are studying is DNA methylation. We hypothesize that the silencing of these genes in most brain areas such as in cortex, striatum, cerebellum, etc, and likely also OT in the SCN, is due to the DNA methylation of these genes in the non-expressing cells. The stable silencing of the OT &VP genes in areas such as cerebral cortex, etc, and possibly of OT in the SCN could be due to DNA methylation, which is much less dynamic and in many cases irreversible. Experiments under way to test this hypothesis use the stereotaxic injection of AAV-LCM strategy developed and described in the summary of project No. 1 Z01 NS002723-24 LNC in order to identify and isolate OT- and VP-MCNs for RNA analysis. However, in this case we isolate DNA from the pools of the individual identified OT- and VP-MCNs for analysis of their methylation patterns by the bisulfate conversion procedure. These experiments require much larger numbers of LCM-isolated MCNs than those directed at RNA analysis by qPCR, and we are presently in the process of collecting the required number of neurons needed for this purpose. Organotypic cultures of mouse and rat magnocellular neurons (MCNs) in the hypothalamo-neurohypophysial system (HNS) and SCN neurons have served as important experimental models for the molecular and physiological study of these neuronal phenotypes. However, it has been difficult to maintain in vivo level numbers of the MCNs, particularly vasopressin MCNs, in these cultures for long periods of time in culture. The vulnerability of the MCNs to axotomy-induced programmed cell death that occurs in organotypic cultures is analogous to the extensive retrograde degeneration of these neurons that occurs in vivo after axonal damage (Shahar et al, 2004). This has led us to the hypothesis that this vulnerability of the MCNs is related to the loss of retrograde trophic factors from the posterior pituitary after axonal injury. Recently, we obtained some support for this hypothesis by employing two LIF-family neurotrophic factors found in the pituitary, leukemia inhibiting factor (LIF) and ciliary neurotrophic factor (CNTF), to rescue rat OT- and VPMCNs from axotomy-induced, programmed cell death in vitro. Quantitative data were obtained that showed the efficacy of the LIF family of neurotrophic factors to enhance the survival of MCNs in three nuclei, the paraventricular (PVN), supraoptic (SON), and accessory (ACC) nuclei in the mouse and rat hypothalamus in vitro (House et al, 2009). This optimization of the survival of the OT- and VP-MCNs in these cultures should greatly facilitate future cell-biological and physiologic studies of these important neuronal systems.